A characteristic feature of many cancer cells is their ability to grow independently of adhesion. In contrast, when untransformed endothelial cells are prevented from adhering to the extracellular matrix (ECM), they undergo apoptosis (Frisch & Francis, 1994; Meredith et al, 1993). The process by which normally adherent cells are triggered to undergo apoptosis when they are unable to adhere to ECM has been termed “anoikis” (Frisch & Ruoslahti, 1997) and is an example of the effect on a cell of removal of a survival factor. Changes in signalling by adhesion molecules can lead to resistance to anoikis (Frisch & Ruoslahti, 1997) and this may contribute to the mechanism whereby cancer cells that grow independently of adhesion are able to avoid anoikis.
Akt (also known as protein kinase B (PKB) or “related to A and C protein kinase” (RAC-PK)) is a serine/threonine kinase that has been implicated in regulating cell survival (Khwaja et al., 1997; Dudek et al., 1997; Kauffmann-Zeh et al., 1997; Kennedy et al., 1997; Datta et al., 1997; Marte & Downward, 1997). Akt can inhibit apoptosis induced by detachment from ECM (Khwaja et al., 1997), as well as by survival factor withdrawal (Kennedy et al., 1997; Ahmed et al., 1997; Dudek et al., 1997; Kauffman-Zeh et al., 1997; Philpott et al., 1997; Crowder & Freeman, 1998; Eves et al., 1998) or irradiation (Kulik et al., 1997).
Akt comprises an NH2-terminal pleckstrin homology (PH) domain involved in lipid binding, a kinase domain and a COOH-terminal “tail”. Akt is thought to be activated by recruitment to the plasma membrane and subsequent phosphorylation by two upstream kinases, PDK-1 and PDK-2 (reviewed in Coffer et al, 1998; Alessi & Cohen, 1998). The binding of 3-phosphoinositides, generated by phosphatidylinositol 3-kinase (PI 3-kinase), to the PH domain of Akt is believed to promote translocation to the plasma membrane and to facilitate phosphorylation of Akt-1 by PDK-1 at Thr308 (Alessi et al., 1996; Alessi et al., 1997; Stephens et al., 1998) or of Akt-2 at Thr309 (Meier et al., 1997). In addition to phosphorylation of Thr3081 full activation requires phosphorylation of the COOH tail at Ser473 in Akt-1 (Alessi et al, 1996) or at Ser474 in Akt-2 (Meier et al., 1997). The enzyme responsible for phosphorylation of Ser473/Ser474 was originally named PDK-2 but recently the integrin-linked kinase, ILK (Delcommenne et al., 1998) has emerged as a candidate for this function.
Two human isoforms of Akt have been described to date, Akt-1 and Akt-2 (Coffer & Woodgett, 1991; Jones et al., 1991; Cheng et al., 1992). A third isoform, here referred to as Akt-3, has been described in the rat (Konishi et al., 1995). Since this rat Akt-3 possesses an apparently truncated tail and thereby lacks Ser473, its regulation may differ from that of Akt-1 and Akt-2. Both Akt-1 and Akt-2 are expressed widely, although the expression of Akt-2 is most prominent in insulin-responsive tissues, such as liver and skeletal muscle (Konishi et al., 1994; Altomare et al., 1995). Akt-1 and Akt-2 are activated by insulin in rat adipocytes, hepatocytes and skeletal muscle. In contrast, Akt-3 does not appear to be strongly activated by insulin in these tissues (Walker et al., 1998). The role of the various Akt isoforms in insulin signalling may limit the utility of compounds that inhibit Akt-1 or Akt-2 activity as such agents may induce symptoms observed in patients with diabetes. We hypothesized that this problem may be avoided by using selective inhibitors of Akt-3 and this prompted us to identify the human analogue of rat Akt-3.